Kumemicinones A-G, Cytotoxic Angucyclinones from a Deep Sea-Derived Actinomycete of the Genus Actinomadura.

Chemical investigation of the fermentation products of a deep sea water-derived actinomycete, Actinomadura sp. KD439, identified seven new angucyclinones, designated as kumemicinones A-G (1-7), together with the known SF2315B and miaosporone E. NMR and MS spectroscopic analyses, combined with X-ray crystallography and quantum chemical calculations of NMR chemical shifts and electronic circular dichroism (ECD) spectra, uncovered the structures of new angucyclinones as regioisomers of SF2315B at the allyl alcohol unit (1 and 2), an epoxy ring-opened γ-hydroxy enone isomer (3), a B/C-ring-rearranged product (4), or dimers with a new mode of bridging (5-7), adding new structural variation to this antibiotic group. The absolute configuration of SF2315B was also determined by comparison of ECD spectra with those of 1 and 2. All the angucyclinones exhibited cytotoxicity against P388 murine leukemia cells, with IC50 values ranging from 1.8 to 53 µM.

Metabolites Produced by a New Lactiplantibacillus plantarum Strain BF1-13 Isolated from Deep Seawater of Izu-Akazawa Protect the Intestinal Epithelial Barrier from the Dysfunction Induced by Hydrogen Peroxide.

This study aimed to investigate the protective effect of the metabolites produced by a new Lactiplantibacillus plantarum strain BF1-13, isolated from deep seawater (DSW), on the intestinal epithelial barrier against the dysfunction induced by hydrogen peroxide (H2O2) and to elucidate the mechanism underlying the effect. Protective effect of the metabolites by strain BF1-13 on the barrier function of the intestinal epithelial model treated with H2O2 was investigated by the transepithelial electrical resistance (TEER). The metabolites enhanced the Claudin-4 (CLDN-4) expression, including at the transcription level, indicated by immunofluorescence staining and quantitative RT-PCR. The metabolites also showed a suppression of aquaporin3 (AQP3) expression. Lactic acid (LA) produced by this strain of homofermentative lactic acid bacteria (LAB) had a similar enhancement on CLDN-4 expression. The metabolites of L. plantarum strain BF1-13 alleviated the dysfunction of intestinal epithelial barrier owing to its enhancement on the tight junctions (TJs) by LA, along with its suppression on AQP3-facilitating H2O2 intracellular invasion into Caco-2 cells. This is the first report on the enhancement of TJs by LA produced by LAB.

Structure Determination, Biosynthetic Origin, and Total Synthesis of Akazaoxime, an Enteromycin-Class Metabolite from a Marine-Derived Actinomycete of the Genus Micromonospora.

A new enteromycin-class antibiotic, akazaoxime (1), possessing an aldoxime functionality in place of O-methyl nitronic acid, was isolated from the cultured extract of a marine-derived actinomycete of the genus Micromonospora, along with known A-76356 (2). The structure of 1, including the absolute stereochemistry of three chiral centers, was established by comprehensive analysis of nuclear magnetic resonance (NMR) and mass spectrometry data coupled with magnetic anisotropy analysis of its phenylglycine methyl ester derivatives. The stereochemistry of 2, not determined previously, was proven to be the same as that of 1 on the basis of the similarity of their NMR and specific rotation data. Precursor feeding experiments using 13C-labeled compounds elucidated that the carbon skeletons of 1 and 2 are constructed from propionate (methylmalonate), leucine, and glycine. Establishment of the concise and flexible synthetic route to 1 enabled us to implement biological evaluation of 1 and its unnatural analogues, demonstrating weak to moderate antimicrobial activities of 1 against Gram-positive Kocuria rhizophila [minimum inhibitory concentration (MIC) of 50 µg/mL] and those of synthetic analogues against a plant pathogen Glomerella cingulata (MIC of 50 µg/mL) and a human pathogen Trichophyton rubrum (MIC of 25-50 µg/mL).

Distribution of class IId bacteriocin-producing Virgibacillus salexigens in various environments.

We performed several experiments using three strains of Virgibacillus salexigens, namely, P2, NT N53, and C-20MoT (DSM 11483T), which were isolated from completely different sources, in relation to bacteriocin production ability. Results of whole-genome sequencing analysis revealed that all strains have very similar sequences encoding class IId bacteriocin. Although a partial amino acid sequence of the purified bacteriocin produced by strain P2 isolated from fermented food was previously reported, whole-genome sequencing and the N-terminal sequencing results in this study showed that its complete amino acid sequence consisted of 48 residues, which corresponded to that of the hypothetical bacteriocin encoded by the gene in Virgibacillus massiliensis strain Vm-5T (DSM 28587T) isolated from the human gut. From the results of 16S rRNA gene sequencing and whole-genome sequencing analyses, we taxonomically confirmed Vm-5T to be a strain of V. salexigens, and its broth culture showed antibacterial activity. Strain NT N53 isolated from the deep-sea floor produced two bacteriocins, namely, NTN-A and NTN-B. The results of N-terminal sequencing, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and whole-genome sequencing analyses showed that their amino acid sequences differed in only one residue, and NTN-A showed the same sequence as the bacteriocin produced by strain P2. Although strain C-20MoT isolated from a solar saltern had the coding sequence very similar to that of NTN-A, its broth culture showed no antibacterial activity. This finding suggests that class IId bacteriocin-producing or bacteriocin-gene-encoding V. salexigens strains are widely distributed in distinct environment sources with different geographical and material properties.

Nomimicins B-D, new tetronate-class polyketides from a marine-derived actinomycete of the genus Actinomadura.

Three new tetronate-class polyketides, nomimicins B, C, and D, along with nomimicin, hereafter named nomimicin A, were isolated from the culture extract of Actinomadura sp. AKA43 collected from floating particles in the deep-sea water of Sagami Bay, Japan. The structures of nomimicins B, C, and D were elucidated through the interpretation of NMR and MS analytical data, and the absolute configuration was determined by combination of NOESY/ROESY and ECD analyses. Nomimicins B, C, and D showed antimicrobial activity against Gram-positive bacteria, Kocuria rhizophila and Bacillus subtilis, with MIC values in the range of 6.5 to 12.5 µg/mL. Nomimicins B and C also displayed cytotoxicity against P388 murine leukemia cells with IC50 values of 33 and 89 µM, respectively.

Streptomyces otsuchiensis sp. nov., a biosurfactant-producing actinobacterium isolated from marine sediment.

A novel actinobacterium producing biosurfactant, designated OTB305T, was isolated from marine sediment sampled at Otsuchi Bay, Iwate Prefecture, Japan and its taxonomic position was examined using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences exhibited that strain OTB305T was closely related to Streptomyces bohaiensis JCM 19630T (98.8 %) and Streptomyces lonarensis DSM 42084T (98.8 %). The chemotaxonomic characteristics of strain OTB305T corresponded to those of the genus Streptomyces as follows: the diamino acid of the cell-wall peptidoglycan was ll-diaminopimelic acid; whole-cell hydrolysates contained glucose and lacked characteristic major sugars; the predominant isoprenoid quinones were MK-9(H8) and MK-9(H6); the polar lipids were phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and an unidentified phospholipid; the major cellular fatty acids were iso-C16 : 0, C16 : 0 and C16 : 1 ω7c; and the genomic DNA G+C content was 72.83 mol%. However, genomic relatedness analysis based on the average nucleotide identity and some phenotypic characteristics revealed that strain OTB305T was distinguished from closely related Streptomyces species. Therefore, strain OTB305T represents a novel species of the genus Streptomyces, for which the name Streptomyces otsuchiensis sp. nov. is proposed. The type strain is OTB305T (=NBRC 113255T=TBRC 9682T).

Konamycins A and B and Rubromycins CA1 and CA2, Aromatic Polyketides from the Tunicate-Derived Streptomyces hyaluromycini MB-PO13T.

Four new aromatic polyketides, konamycins A (1) and B (2) and rubromycins CA1 (3) and CA2 (4), were isolated from the culture extract of the tunicate-derived Streptomyces hyaluromycini MB-PO13T. Compounds 1 and 2 possess a benzo[ b]fluorene aglycon modified by C-glycosylation with l-amicetose. Compounds 3 and 4 are the new congeners of rubromycin in which a naphthoquinone and carboxylated isocoumarin are joined through a spiroketal carbon. The structures of these compounds were determined by extensive analysis of 1D and 2D NMR spectroscopic data. Compound 1 showed radical scavenging activity in DPPH and superoxide quenching assays, and 3 and 4 displayed antimicrobial activity against Gram-positive bacteria.

Lysinimicrobium sediminis sp. nov., an actinobacterium isolated from estuary sediment.

A novel Gram-stain-positive actinobacterium, designated HT7-17T, was isolated from a sediment sample collected from the estuary of the Tama River, Japan, and its taxonomic position was investigated using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequence comparisons revealed that strain HT7-17T was closely related to members of the genus Lysinimicrobium, with a similarity range of 97.1-98.2 %. The peptidoglycan type of strain HT7-17T was A4α, the predominant menaquinone was demethylmenaquinone DMK-9(H4) and the major fatty acids were anteiso-C15 : 0, C16 : 0 and iso-C16 : 0. The DNA G+C content was 69.9 mol%. These chemotaxonomic features corresponded to those of the genus Lysinimicrobium. Meanwhile, the differences in some phenotypic characteristics, along with the result of DNA-DNA hybridization, indicated that strain HT7-17T should be distinguished from the recognized species of the genus Lysinimicrobium. Therefore, strain HT7-17T represents a novel species of the genus Lysinimicrobium, for which the name Lysinimicrobium sediminis sp. nov. is proposed. The type strain is HT7-17T (=NBRC 112286T=TBRC 7037T).

Diversity of the bacterial community in Myanmar traditional salted fish yegyo ngapi.

The distribution and characterization of bacteria including lactic acid bacteria (LAB) in the traditional and popular salted fish yegyo ngapi in Myanmar were studied to clarify the contribution of these bacteria to the curing and ripening of this product. Samples of yegyo ngapi purchased from a market in Yangon were used. Most of the isolates obtained using de Man, Rogosa and Sharpe medium containing 10 % NaCl were identified as coccoid LAB on the basis of their basic phenotypic characteristics. From the results of 16S rRNA gene sequencing and PCR-restriction fragment length polymorphism analysis of this gene, most of the isolates were identified as the halophilic LAB Tetragenococcus muriaticus. Analyses of the 16S rRNA gene based on the clone library using DNA extracted from salted fish products were also performed. The results of these molecular-analysis-based techniques showed that spore-forming and non-spore-forming anaerobic bacteria including the genera Clostridium and Halanaerobium in addition to T. muriaticus were also frequently found in bacterial communities. These findings suggest that the anaerobic condition during curing and ripening resulted in bacterial communities composed of strictly anaerobic bacteria and halophilic LAB, and that these bacteria might also contribute to the manufacturing processes of this product. In addition, DNA sequences similar to that of Clostridium botulinum were found in the clone library analysis. Therefore, despite no reports of botulism poisoning from the region where the samples were taken, closer surveillance should be carried out from the viewpoint of food safety.

Production of bacteriocin by Virgibacillus salexigens isolated from "terasi": a traditionally fermented shrimp paste in Indonesia.

A natural antibacterial-substance-producing gram-positive bacterium was isolated from terasi shrimp paste, a popular fermented product in Indonesia. This strain, a spore-forming and strictly aerobic bacterium, was identified as Virgibacillus salexigens by 16S rRNA gene sequence analysis. The antibacterial substance purified from the precipitated product in the culture supernatant of the strain using ammonium sulfate showed a broad inhibition spectrum against gram-positive bacteria, including a typical foodborne bacterium, namely, Listeria monocytogenes. The antibacterial activity of the substance was inactivated by treatments with various proteolytic enzymes. It was stable after heating or pH treatment, and approximately 60% of the initial activity remained even after heating at 121 °C for 15 min. In addition, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis indicated that its monoisotopic mass weight was 5318.4 Da (M+H)(+). On the basis of the results obtained by the automated Edman degradation technique and MALDI-TOF MS analysis, the substance can be classified as a member of Class IId bacteriocins, but it could not be identified as any of the previously purified substances except for the putative bacteriocin predicted from the draft genome sequence data of gram-positive bacteria such as Virgibacillus and Bacillus strains.

Hyaluromycin, a new hyaluronidase inhibitor of polyketide origin from marine Streptomyces sp.

Hyaluromycin (1), a new member of the rubromycin family of antibiotics, was isolated from the culture extract of a marine-derived Streptomyces sp. as a HAase inhibitor on the basis of HAase activity screening. The structure of 1 was elucidated through the interpretation of NMR data for the compound and its 3″-O-methyl derivative in combination with an incorporation experiment with [1,2-13C2]acetate. The compound's absolute configuration was determined by the comparison of its circular dichroism (CD) spectrum with those of other rubromycins. Hyaluromycin (1) consists of a γ-rubromycin core structure possessing a 2-amino-3-hydroxycyclopent-2-enone (C5N) unit as an amide substituent of the carboxyl function; both structural units have been reported only from actinomycetes. Hyaluromycin (1) displayed approximately 25-fold more potent hyaluronidase inhibitory activity against hyaluronidase than did glycyrrhizin, a known inhibitor of plant origin.

Nocapyrones: α- and γ-pyrones from a marine-derived Nocardiopsis sp.

One new α-pyrone (nocapyrone R (1)), and three known γ-pyrones (nocapyrones B, H and L (2-4)) were isolated from the culture extract of a Nocardiopsis strain collected from marine sediment. Structures of these compounds were determined on the basis of spectroscopic data including NMR and MS. γ-Pyrones 2-4 were found to induce adiponectin production in murine ST-13 preadipocyte cells but the α-pyrone 1 had no activity. The absolute configuration of the anteiso-methyl branching in 4 was determined by HPLC comparison of a degraded product of 4 with standard samples as a 2:3 enantiomeric mixture of (R)- and (S)-isomers.

A thermophilic, hydrogenogenic and carboxydotrophic bacterium, Calderihabitans maritimus gen. nov., sp. nov., from a marine sediment core of an undersea caldera.

A hydrogenogenic, carboxydotrophic marine bacterium, strain KKC1(T), was isolated from a sediment core sample taken from a submerged marine caldera. Cells were non-motile, Gram-stain-negative, 1.0-3.0 µm straight rods, often observed with round endospores. Strain KKC1(T) grew at 55-68 °C, pH 5.2-9.2 and 0.8-14 % (w/v) salinity. Optimum growth occurred at 65 °C, pH 7.0-7.5 and 2.46 % salinity with a doubling time of 3.7 h. The isolate grew chemolithotrophically, producing H2 from carbon monoxide (CO) oxidation with reduction of various electron acceptors, e.g. sulfite, thiosulfate, fumarate, ferric iron and AQDS (9,10-anthraquinone 2,6-disulfonate). KKC1(T) grew heterotrophically on pyruvate, lactate, fumarate, glucose, fructose and mannose with thiosulfate as an electron acceptor. When grown mixotrophically on CO and pyruvate, C16 : 0 constituted almost half of the total cellular fatty acids. The DNA G+C content was 50.6 mol%. The 16S rRNA gene sequence of KKC1(T) was most closely related to those of members of the genus Moorella with similarity ranging from 91 to 89 %. Based on physiological and phylogenetic novelty, we propose the isolate as a representative of a new genus and novel species with the name Calderihabitans maritimus gen. nov., sp. nov.; the type strain of the type species is KKC1(T) ( = DSM 26464(T) = NBRC 109353(T)).

An effective method based on wet-heat treatment for the selective isolation of Micromonospora from estuarine sediments.

Several methods for the isolation of Micromonospora from soil samples have been developed; however, it is unclear whether these methods are optimal for estuarine samples. In this study, we optimized the conditions of a wet-heat method for the selective isolation of Micromonospora from estuarine sediments. Sediments were collected from the Arakawa River (estuarine sediments) and Tokyo Bay (marine sediments). Sediment samples were wet-heated at 45, 55, or 65 °C for 30 min and then incubated at 27 °C for 3 weeks. After incubation, most of the actinomycete colonies were macroscopically determined to be of the genus Micromonospora or Streptomyces. In contrast to the treatment at 55 °C, treatment at 65 °C drastically reduced the number of Streptomyces colonies but increased the number of Micromonospora colonies from the estuarine sediments. This procedure allowed us to grow cultures that were composed of more than 90 % Micromonospora. In addition, treatment at 65 °C did not affect the diversity of Micromonospora species compared with treatment at 55 °C. These results indicate that the wet-heat method, which involves pre-treating the sediment at 65 °C for 30 min, is a very simple and effective method for the selective enrichment of a large number of diverse Micromonospora from estuarine sediments. Our results may lead to the isolation of new Micromonospora species, which produce novel bioactive compounds, from different estuarine sediments.

Bacterial production of the tunicate-derived antitumor cyclic depsipeptide didemnin B.

Natural products obtained from marine invertebrates such as sponges and tunicates are attractive sources of drugs. However, a critical obstacle in the development of these compounds is the problem of supply. In most cases, neither chemical synthesis nor mariculture of invertebrates is economically feasible. Due to structural similarities, many marine natural products are suspected to be produced by associated microorganisms. A favorable strategy for the production of such compounds is to use culturable microorganisms. Here we report that didemnin B, a tunicate-derived depsipeptide, has been isolated from a culturable bacterium, Tistrella mobilis YIT 12409.

Development of an in vivo-mimic silkworm infection model with Mycobacterium avium complex.

In vivo-mimic silkworm infection models with Mycobacterium avium and Mycobacterium intracellulare were newly established to evaluate the therapeutic effects of anti-M. avium complex (MAC) antibiotics. Silkworms raised at 37°C died within 72 hours of an injection of M. avium or M.intracellulare (2.5 × 107 colony-forming unit (CFU)/larva·g) into the hemolymph. Clinical anti-mycobacterial (tuberculosis) antibiotics were evaluated under these conditions. Clarithromycin, kanamycin, streptomycin, amikacin, and ciprofloxacin exerted therapeutic effects in a dose-dependent manner, which was consistent with those in the mouse model. Furthermore, three effective actinomycete culture broths were selected in the screening program of our microbial broth library using the silkworm model, and four active metabolites, ohmyungsamycins A and B (1 and 2), chartreusin (3), and griseoviridin (4), were identified. Among these compounds, 1 showed the lowest 50% effective dose (ED50) value (8.5 µg/larva·g), while 3 had the best ED50/minimum inhibitory concentration (MIC) ratio (7.4). These results indicate that silkworm models are a useful tool for identifying anti-MAC antibiotics candidates with veritable therapeutic effects.

A new diketopiperazine-like inhibitor of bone morphogenetic protein-induced osteoblastic differentiation produced by marine-derived Aspergillus sp. BFM-0085.

A new diketopiperazine-like compound, designated protuboxepin K (1), was isolated together with the known structurally related protuboxepin A (2) from culture broth of the marine-derived fungal strain Aspergillus sp. BFM-0085 isolated from a sediment sample of Tokyo Bay. The structure of protuboxepin K was elucidated by spectroscopic data, including 1D and 2D NMR. Compounds 1 and 2 inhibited bone morphogenetic protein (BMP)-induced alkaline phosphatase activity with IC50 values of 4.7 and 25.2 µM, respectively, in mutant BMP receptor-carrying C2C12(R206H) cells.

Diversity of salt-tolerant tellurate-reducing bacteria in a marine environment.

Tellurium (Te) has been increasingly used as a semiconductor material in copious amounts, with a concomitant increase in its discharge from industrial effluents and mining wastewater into the environment. However, soluble Te, such as tellurate (VI) and tellurite (IV), is toxic to organisms. Thus, highly efficient technologies need to be developed for a double-benefit detoxification and recovery of soluble Te from industrial and mining wastewater. Since industrial wastewater contains high concentrations of salt, salt-tolerant microorganisms that metabolize rare metals such as Te have been the subject of focus for the effective detoxification and recovery of Te. In the present study, a total of 52 salt-tolerant tellurate-reducing microorganisms were isolated from marine environmental samples. Of these, 18 strains achieved greater than, or equal to, 50% removal of water-soluble Te from a medium containing 0.4 mM tellurate after 72 h incubation. The 18 isolated strains belonged to 13 species of the following 9 genera: Sulfitobacter, Ruegeria, Hoeflea, Alteromonas, Marinobacter, Pseudoalteromonas, Shewanella, Idiomarina, and Vibrio. No microorganism has been reported to reduce tellurate and tellurite from six of the aforementioned genera, namely, Sulfitobacter, Ruegeria, Alteromonas, Marinobacter, Idiomarina, and Vibrio. Especially, one of the isolates Sulfitobacter sp. strain TK39B, removed 82% (w/w) of soluble Te with a 4% NaCl tolerance. These results showed that salt-tolerant tellurate-reducing bacteria that can be used in the detoxification and recovery of Te are widely present in the marine environment.

Akazamicin, a cytotoxic aromatic polyketide from marine-derived Nonomuraea sp.

In our screening program on marine-derived actinomycetes, Nonomuraea sp. AKA32 isolated from deep-sea water collected from a depth of 800 m in Sagami Bay, Japan was found to produce compounds cytotoxic to cancer cells. Activity-guided purification led to the isolation of a new aromatic polyketide, akazamicin (1), along with two known compounds, actinofuranone C (2) and N-formylanthranilic acid (3). Structures of these compounds were elucidated through the interpretation of NMR and MS spectroscopic data. Compounds 1, 2, and 3 displayed cytotoxicity against murine B16 melanoma cell line with the IC50 value of 1.7, 1.2, and 25 µM, respectively.

Actinomycetal community structures in seawater and freshwater examined by DGGE analysis of 16S rRNA gene fragments.

The actinomycetal community structures in marine and freshwater environments (the Pacific Ocean, East China Sea, Tokyo Bay, and Arakawa River) were investigated by a culture-independent molecular method to clarify spatial and seasonal distributions. Deoxyribonucleic acid (DNA) was extracted from environmental water samples, and a community analysis was carried out on polymerase chain reaction-amplified 16S ribosomal DNA. The amplified DNA fragments were analyzed by denaturing gradient gel electrophoresis (DGGE) and nonmetric multidimensional scaling analysis, followed by sequencing analysis. The actinomycetal community structures were different at each station in the Pacific Ocean, the East China Sea, Tokyo Bay, and Arakawa River, and different populations predominated in each area. There were vertical variations in actinomycetal communities in the Pacific Ocean and East China Sea between the surface and 100-m depth, but communities were similar from 200- to 1,000-m depths. There were also distinct seasonal variations in communities in Tokyo Bay. Phylogenetic analysis of DNA fragments recovered from DGGE bands revealed that most of the predominant actinomycetal strains were uncultured and were quite different from well known culturable strains, such as the Streptomyces, Micromonospora, Microbispora, Salinispora, and Actinoplanes groups. These results suggest that the marine environment is an attractive target for discovering new actinomycetal populations producing bioactive compounds and that sampling depth and season are important considerations for isolating various populations effectively.